Fungal transcriptional activator useful in methods for producing polypeptides

ABSTRACT

The present invention relates to isolated nucleic acid sequences encoding polypeptides having transcriptional activation activity and to the polypeptides. The invention also relates to nucleic acid constructs, vectors and host cells comprising the nucleic acid sequences. The invention further relates to host cells useful for the production of polypeptides in which the production or function of the transcriptional activator has been altered, as well as to methods for producing the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/525,305filed Mar. 14, 2000, now U.S. Pat. No. 6,806,062, which is acontinuation-in-part of U.S. application Ser. No. 09/411,925 filed Oct.4, 1999, now abandoned which claims priority or the benefit under 35U.S.C. 119 of Danish Application No. PA 1998 01258 filed Oct. 5, 1998and U.S. Provisional Application No. 60/103,945 filed Oct. 13, 1998, thecontents of which are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated nucleic acid sequencesencoding polypeptides having transcriptional activation activity and tothe polypeptides. The invention also relates to nucleic acid constructs,vectors and host cells comprising the nucleic acid sequences. Theinvention further relates to host cells useful for the production ofpolypeptides in which the production or function of the transcriptionalactivator has been altered, as well as to methods for producing thepolypeptides.

2. Description of the Related Art

The use of recombinant host cells in the expression of heterologousproteins has in recent years greatly simplified the production of largequantities of commercially valuable proteins which otherwise areobtainable only by purification from their native sources. Currently,there is a varied selection of expression systems from which to choosefor the production of any given protein, including eubacterial andeukaryotic hosts. The selection of an appropriate expression systemoften depends not only on the ability of the host cell to produceadequate yields of the protein in an active state, but, to a largeextent, may also be governed by the intended end use of the protein.

One problem frequently encountered is the high level of proteolyticenzymes produced by a given host cell or present in the culture medium.One suggestion has been to provide host organisms deprived of theability to produce specific proteolytic compounds. For example, WO90/00192 (Genencor, Inc.) describes filamentous fungal hosts incapableof secreting enzymatically active aspartic proteinase. EP 574 347 (CibaGeigy AG) describes Aspergillus hosts defective in a serine protease ofthe subtilisin-type. WO 98/12300 (Novo Nordisk A/S) describes hostsdefective in a metalloprotease and an alkaline protease. WO 97/12045(Genencor, Inc.) describes yeast and bacterial host systems, which arerendered protease deficient resulting from a disruption of a promotersequence involved in the regulation of a protease gene.

Mattern, I. E., et al., (1992. Mol Gen Genet 234:332-336) describe amutant strain of Aspergillus niger, which was shown to have only 1 to 2%of the extracellular protease activity of the parent strain, apparentlydue to a deficiency of at least two proteases, aspergillopepsin A andaspergillopepsin B. It was suggested that the protease deficientphenotype could result from a regulatory mutation affecting theexpression of the genes coding for both proteases.

The initiation of eukaryotic transcription at a specific promoter or setof promoters requires a eukaryotic transcriptional activator which is apolypeptide, but which is not itself part of RNA polymerase. Manytranscriptional activators bind to a specific site on the promoter toform a functional promoter necessary for the initiation of transcriptionof the polypeptide encoding sequence. However, a transcriptionalactivator may also be incorporated into an initiation complex only inthe presence of other polypeptides. Polypeptides with transcriptionalactivation activity have been described in fungi, and a list of suchpolypeptides has been published (Dhawale, S. S., and Lane, A. C. 1993.Nucleic Acid Research 21:5537-5546).

It is an object of the present invention to provide improved methods forincreasing production of polypeptides in host cells in which theactivity of a transcriptional activator involved in the regulation ofprotease production has been modified.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to an isolated nucleicacid sequence encoding a transcriptional activator selected from thegroup consisting of:

(a) a nucleic acid sequence having at least 70% identity with thenucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 48;

(b) a nucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least 50% identity with the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 49;

(c) a nucleic acid sequence which hybridizes under low stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 1 or SEQ IDNO: 48, or (ii) its complementary strand, wherein the low stringencyconditions are defined by prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 microg/ml sheared and denatured salmon spermDNA, and 25% formamide, and wash conditions are defined by 50° C. for 30minutes in 2×SSC, 0.2% SDS;

(d) an allelic variant of (a), (b), or (c);

(e) a subsequence of (a), (b), (c), or (d), wherein the subsequenceencodes a polypeptide fragment which has transcriptional activationactivity; and

(f) a subsequence of (a), (b) (c), or (d), wherein the subsequenceencodes a polypeptide with the amino acid sequence of SEQ ID NO: 3.

The nucleic acid sequence shown in SEQ ID NO: 1 is the Aspergillus nigerprtT gene encoding the transcriptional factor shown in SEQ ID NO: 2 asdescribed further below and in the Examples.

The nucleic acid sequence shown in SEQ ID NO: 48 is the Aspergillusoryzae IFO4177 prtT gene encoding the transcriptional factor shown inSEQ ID NO: 49. The A. oryzae prtT gene has a coding region starting inposition 795 and ending at position 2931. The prtT gene has 4 introns inpositions 1028-1135, 1538-1591, 2018-2066, and 2297-2347, respectively.This is described further below and in the Examples.

In another aspect, the invention also relates to nucleic acidconstructs, vectors and host cells comprising the nucleic acidsequences, and to the polypeptides encoded by the nucleic acidsequences. The invention further relates to host cells useful for theproduction of a polypeptide, in which the production or function of thetranscriptional activator has been altered, as well as to methods forproducing the polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of the plasmid pPAP, the construction ofwhich is described in Example 1.

FIG. 2 shows a restriction map of the plasmid pAopyrGcosArp1, theconstruction of which is described in Example 1.

FIG. 3 shows a restriction map of the plasmid pEES1, the construction ofwhich is described in Example 1.

FIG. 4 shows a restriction map of the plasmid pDprt, the construction ofwhich is described in Example 3.

FIG. 5 shows a restriction map of the plasmid pGPprt, the constructionof which is described in Example 4.

FIG. 6 shows the sequence of the insert in the two plasmids containingthe PCR fragment of the A. oryzae prtT Zn²⁺-finger. ICA217 is thesequence from one of the plasmids and ICA218 is the sequence from theother.

FIG. 7 shows plasmid pDV8 a pSP65 (Promega™) based plasmid containingthe HSV-tk gene on a 1.2 kb BglII/BamHI fragment inserted between a 1.0kb XhoI/BglII fragment of the A. nidulans gpd promoter and a 0.8 kbBamHI/HindIII fragment containing the A. nidulans trpC transcriptionalterminator.

FIG. 8 shows the construction of pJaL554 described in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic Acid Sequences Encoding Transcriptional Activators

A first aspect of the present invention relates to an isolated nucleicacid sequence encoding a transcriptional activator selected from thegroup consisting of:

(a) a nucleic acid sequence having at least 70% identity with thenucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 48;

(b) a nucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least 50% identity with the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 49;

(c) a nucleic acid sequence which hybridizes under low stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 1 or SEQ IDNO: 48, or (ii) its complementary strand, wherein the low stringencyconditions are defined by prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micro g/ml sheared and denatured salmon spermDNA, and 25% formamide, and wash conditions are defined by 50° C. for 30minutes in 2×SSC, 0.2% SDS;

(d) an allelic variant of (a), (b), or (c);

(e) a subsequence of (a), (b), (c), or (d), wherein the subsequenceencodes a polypeptide fragment which has transcriptional activationactivity; and

(f) a subsequence of (a), (b), (c), or (d), wherein the subsequenceencodes a polypeptide with the amino acid sequence of SEQ ID NO: 3.

The term “transcriptional activator” as used herein refers to apolypeptide which has the capability to activate a specific promoter orset of promoters necessary for the initiation of transcription of thepolypeptide encoding sequence to which it is linked.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

In a preferred embodiment, the nucleic acid sequence has a degree ofidentity to the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQID NO: 48 of at least about 70%, preferably at least about 80%, morepreferably at least about 85%, even more preferably at least about 90%,most preferably at least about 95%, even most preferably at least about97%, and even more preferred at least 99% identity, which encodes anactive polypeptide. For purposes of the present invention, the degree ofidentity between two nucleic acid sequences is determined by the Clustalmethod (Higgins, 1989, CABIOS 5:151-153) with an identity table, a gappenalty of 10, and a gap length penalty of 10.

In an even more preferred embodiment, the nucleic acid sequence encodinga transcriptional activator has a nucleic acid sequence as set forth inSEQ ID NO: 1 or SEQ ID NO: 48.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source. For example, itmay be of interest to synthesize variants of the polypeptide where thevariants differ in specific activity, binding specificity and/oraffinity, or the like using, e.g., site-directed mutagenesis. Theanalogous sequence may be constructed on the basis of the nucleic acidsequence presented as the polypeptide encoding part of SEQ ID NO: 1 orSEQ ID NO: 48, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich corresponds to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

In another preferred embodiment, the present invention relates toisolated nucleic acid sequences encoding polypeptides having an aminoacid sequence which has a degree of identity to the amino acid sequenceset forth in SEQ ID NO: 2 or SEQ ID NO: 49 of at least about 50%,preferably at least about 60%, preferably at least about 70%, morepreferably at least about 80%, even more preferably at least about 90%,most preferably at least about 95%, even most preferably at least about97%, and even more preferred at least 99%, which qualitatively retainthe transcriptional activation activity of the polypeptides (hereinafter“homologous polypeptides”).

In a preferred embodiment, the homologous polypeptides have an aminoacid sequence which differs by five amino acids, preferably by fouramino acids, more preferably by three amino acids, even more preferablyby two amino acids, and most preferably by one amino acid from the aminoacid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 49. For purposesof the present invention, the degree of identity between two amino acidsequences is determined by the Clustal method (Higgins, 1989, supra)with an identity table, a gap penalty of 10, and a gap length penalty of10.

Hybridization indicates that by methods of standard Southern blottingprocedures, the nucleic acid sequence hybridizes to an oligonucleotideprobe corresponding to the polypeptide encoding part of the nucleic acidsequence shown in SEQ ID NO: 1, under low to high stringency conditions(i.e., prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS,200 micrograms/ml sheared and denatured salmon sperm DNA, and either 25,35 or 50% formamide for low, medium and high stringencies,respectively). In order to identify a clone or DNA which is homologouswith SEQ ID NO: 1 or SEQ ID NO: 48, the hybridization reaction is washedthree times for 30 minutes each using 2×SSC, 0.2% SDS preferably atleast 50° C., more preferably at least 55° C., more preferably at least60° C., more preferably at least 65° C., even more preferably at least70° C., and most preferably at least 75° C.

The nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 48, or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 49, or a partial sequence thereof, or the amino acidsequence of SEQ ID NO: 3, may be used to design an oligonucleotide probeto identify and isolate or clone a homologous gene of any genus orspecies according to methods well known in the art.

In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 40 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). For example, molecules to which a³²P—, ³H— or ³⁵S-labelled oligonucleotide probe hybridizes may bedetected by use of X-ray film.

Thus, a genomic, cDNA or combinatorial chemical library prepared fromsuch other organisms may be screened for DNA which hybridizes with theprobes described above and which encodes a polypeptide withtranscriptional activation activity. Genomic or other DNA from suchother organisms may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. A clone or DNA whichis homologous to SEQ ID NO: 1 or SEQ ID NO: 48 may then be identifiedfollowing standard Southern blotting procedures.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chomosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (i.e., no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences.

The term “allelic variant of a polypeptide” is a polypeptide encoded byan allelic variant of a gene. In a preferred embodiment, the nucleicacid sequence encoding a transcriptional activator of the presentinvention is an allelic variant of a nucleic acid sequence selected fromthe group consisting of nucleic acid sequences: (a) having at least 70%identity with the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:48, (b) encoding a polypeptide having an amino acid sequence which hasat least 50% identity with the amino acid sequence of SEQ ID NO: 2 orSEQ ID NO: 49, (c) which hybridizes under low stringency conditions withthe nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 48, or itscomplementary strand, and (d) encoding a polypeptide having the aminoacid sequence of SEQ ID NO: 3.

The present invention also encompasses nucleic acid sequences whichdiffer from SEQ ID NO: 1 or SEQ ID NO: 48 by virtue of the degeneracy ofthe genetic code. The present invention also relates to subsequences ofSEQ ID NO: 1 or SEQ ID NO: 49, wherein a subsequence of SEQ ID NO: 1 isa nucleic acid sequence encompassed by SEQ ID NO: 1 or SEQ ID NO: 48except that one or more nucleotides from the 5′ and/or 3′ end have beendeleted. Preferably, a subsequence of SEQ ID NO: 1 or SEQ ID NO: 48encodes a polypeptide fragment which has transcriptional activationactivity. In a more preferred embodiment, a subsequence of SEQ ID NO: 1or SEQ ID NO: 48 contains at least a nucleic acid sequence encoding thepolypeptide sequence shown in SEQ ID NO: 3.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using methods based on polymerase chainreaction (PCR) or antibody screening of expression libraries to detectcloned DNA fragments with shared structural features. (See, e.g., Inniset al., 1990, PCR: A Guide to Methods and Application, Academic Press,New York.) Other nucleic acid amplification procedures such as ligasechain reaction (LCR), ligated activated transcription (LAT) and nucleicacid sequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a microorganism, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleic acid sequence.

The transcriptional activators encoded by nucleic acid sequences whichhybridize with an oligonucleotide probe which hybridizes with thenucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 48, itscomplementary strand, or allelic variants and subsequences of SEQ ID NO:1 or SEQ ID NO: 48, or allelic variants and fragments of thetranscriptional activators may be obtained from microorganisms of anygenus.

In a preferred embodiment, the transcriptional activators may beobtained from a fungal cell. “Fungi” as used herein includes the phylaAscomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as definedby Hawksworth, et al., in Ainsworth and Bisby's Dictionary of The Fungi,8th edition, 1995, CAB International, University Press, Cambridge, UK)as well as the Oomycota (as cited in Hawksworth et al., 1995, supra,page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

In preferred embodiment, the fungal source is a filamentous fungalstrain. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelia wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. Filamentous fungal strainsinclude, but are not limited to, strains of Acremonium, Aspergillus,Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces,Thermoascus, Thielavia, Tolypocladium, and Trichoderma.

In a more preferred embodiment, the nucleic acid sequence encoding atranscriptional activator of the present invention is obtained from astrain of Aspergillus, such as A. awamori or A. nidulans. Preferably,the nucleic acid sequence is obtained from a strain of A. niger or A.oryzae. Even more preferably, the nucleic acid sequence is obtained froman isolate of a strain of A. niger, DSM 12298; e.g., the nucleic acidsequence set forth in SEQ ID NO: 1, or from A. oryzae IFO 4177, i.e.,the nucleic acid sequence set forth in SEQ ID NO: 48.

In another more preferred embodiment, the nucleic acid sequence encodinga transcriptional activator of the present invention is obtained from astrain of Fusarium, such as F. oxysporum. Preferably, the strain is astrain of F. venenatum (Nirenberg sp. nov.).

In another preferred embodiment, the nucleic acid sequence encoding atranscriptional activator of the present invention is obtained from ayeast strain, such as a Candida, Kluyveromyces, Schizosaccharomyces, orYarrowia strain. Preferably, the strain is a strain of Hansenula,Pichia, or Saccharomyces.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents. For example, the polypeptides maybe obtained from microorganisms which are taxonomic equivalents ofAspergillus as defined by Raper, K. D. and Fennel, D. I. (1965. TheGenus Aspergillus, The Wilkins Company, Baltimore Md.), regardless ofthe species name by which they are known. Aspergilli are mitosporicfungi characterized by an aspergillum comprised of a conidiospore stipewith no known teleomorphic states terminating in a vesicle, which inturn bears one or two layers of synchronously formed specialized cells,variously referred to as sterigmata or phialides, and asexually formedspores referred to as conidia. Known teleomorphs of Aspergillus includeEurotium, Neosartorya, and Emericella. Strains of Aspergillus andteleomorphs thereof are readily accessible to the public in a number ofculture collections, such as the American Type Culture Collection(ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSM), Centraalbureau Voor Schimmelcultures (CBS), and AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter (NRRL).

Furthermore, such transcriptional activators may be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) using the above-mentionedprobes. Techniques for isolating microorganisms from natural habitatsare well known in the art. The nucleic acid sequence may then be derivedby similarly screening a genomic or cDNA library of anothermicroorganism. Once a nucleic acid sequence encoding a transcriptionalactivator has been detected with the probe(s), the sequence may beisolated or cloned by utilizing techniques which are known to those ofordinary skill in the art (see, e.g., J. Sambrook, E. F. Fritsch, and T.Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.).

In another preferred embodiment, the isolated nucleic acid sequenceencodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 49, or a fragment thereof, which has transcriptionalactivation activity.

In another preferred embodiment, the isolated nucleic acid sequenceencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3.

The present invention also relates to isolated nucleic acid sequencesencoding a transcriptional activator of the present invention, which,e.g., using methods of standard Southern blotting procedures describedabove (cf., Sambrook, et al., 1989, supra), hybridize under lowstringency conditions, more preferably medium stringency conditions, andmost preferably high stringency conditions, with an oligonucleotideprobe which hybridizes under the same conditions with the nucleic acidsequence set forth in SEQ ID NO: 1 or SEQ ID NO: 48 or its complementarystrand, or allelic variants and subsequences of SEQ ID NO: 1 or SEQ IDNO: 48 which encode polypeptide fragments which are transcriptionalactivators in fungi.

In another more preferred embodiment, the nucleic acid sequence is thenucleic acid sequence encoding a polypeptide, which has DNA bindingactivity contained in the plasmid pEES which is contained in Escherichiacoli DSM 12294.

Nucleic Acid Constructs

Another aspect of the present invention relates to nucleic acidconstructs comprising a nucleic acid sequence encoding a transcriptionalactivator of the present invention operably linked to one or morecontrol sequences, which direct the production of the transcriptionalactivator in a suitable expression host. In a preferred embodiment, thenucleic acid sequence encodes a polypeptide, which is contained in theplasmid pEES harboured in Escherichia coli DSM 12294, or the nucleicacid sequence shown in SEQ ID NO: 48 encoding the polypeptide shown inSEQ ID NO: 49.

Expression will be understood to include any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion. Manipulation of thenucleic acid sequence encoding a polypeptide prior to its insertion intoa vector may be desirable or necessary depending on the expressionvector. The techniques for modifying nucleic acid sequences utilizingcloning methods are well known in the art.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence. The term “coding sequence” as defined herein is a sequence,which is transcribed into mRNA and translated into a transcriptionalactivator of the present invention. The boundaries of the codingsequence are generally determined by the ATG start codon at the 5′ endof the mRNA and a transcription terminator sequence located justdownstream of the open reading frame at the 3′ end of the mRNA. A codingsequence can include, but is not limited to, DNA, cDNA, and recombinantnucleic acid sequences.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolypeptide. Each control sequence may be native or foreign to thenucleic acid sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, a polyadenylation sequence, apropeptide sequence, a promoter, a signal sequence, and a transcriptionterminator. At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals. The control sequencesmay be provided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the control sequences withthe coding region of the nucleic acid sequence encoding a polypeptide.The term “operably linked” is defined herein as a configuration in whicha control sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the production of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence, which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence, which shows transcriptionalactivity in the cell including mutant, truncated, and hybrid promoters,and may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the cell.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a filamentous fungal cell toterminate transcription. The terminator sequence is operably linked tothe 3′ terminus of the nucleic acid sequence encoding the polypeptide.Any terminator, which is functional in the cell, may be used in thepresent invention.

Preferred terminators for filamentous fungal cells are obtained from thegenes encoding Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by thefilamentous fungal cell. The leader sequence is operably linked to the5′ terminus of the nucleic acid sequence encoding the polypeptide. Anyleader sequence, which is functional in the cell, may be used in thepresent invention.

Preferred leaders for filamentous fungal cells are obtained from thegenes encoding Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the filamentous fungalcell as a signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence, which is functional in the cell, may be usedin the present invention.

Preferred polyadenylation sequences for filamentous fungal cells areobtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase.

The control sequence may also be a signal peptide-coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide, which can direct the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide-coding region naturallylinked in translation reading frame with the segment of the codingregion, which encodes the secreted polypeptide. Alternatively, the 5′end of the coding sequence may contain a signal peptide-coding region,which is foreign to the coding sequence. The foreign signalpeptide-coding region may be required where the coding sequence does notnormally contain a signal peptide-coding region. Alternatively, theforeign signal peptide-coding region may simply replace the naturalsignal peptide-coding region in order to obtain enhanced secretion ofthe polypeptide. The signal peptide-coding region may be obtained from aglucoamylase or an amylase gene from an Aspergillus species, or a lipaseor proteinase gene from a Rhizomucor species. However, any signalpeptide-coding region, which directs the expressed polypeptide into thesecretory pathway of a filamentous fungal cell, may be used in thepresent invention.

An effective signal peptide coding region for filamentous fungal cellsis the signal peptide coding region obtained from the Aspergillus oryzaeTAKA amylase gene, Aspergillus niger neutral amylase gene, Rhizomucormiehei aspartic proteinase gene, or Humicola lanuginosa cellulase gene.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theRhizomucor miehei aspartic proteinase gene, or the Myceliophthorathermophila laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence encoding the polypeptide may beexpressed by inserting the sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence encoding the polypeptide. The choice of the vector willtypically depend on the compatibility of the vector with the filamentousfungal cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e., a vector, which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the filamentous fungal cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. The vector system maybe a single vector or plasmid or two or more vectors or plasmids, whichtogether contain the total DNA to be introduced into the genome of thefilamentous fungal cell, or a transposon.

The vectors preferably contain one or more selectable markers, whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like. Aselectable marker for use in a filamentous fungal cell may be selectedfrom the group including, but not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents from other species. Preferred for use in an Aspergillus cellare the amdS and pyrG genes of Aspergillus nidulans or Aspergillusoryzae and the bar gene of Streptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits stableintegration of the vector into the host cell genome or autonomousreplication of the vector in the cell independent of the genome of thecell.

Host Cells

Another aspect of the present invention relates to host cells comprisinga nucleic acid construct or an expression vector of the presentinvention.

The choice of a host cell in the methods of the present invention willto a large extent depend upon the source of the nucleic acid sequenceencoding the polypeptide of interest.

The introduction of an expression vector or a nucleic acid constructinto a filamentous fungal cell may involve a process consisting ofprotoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se. Suitableprocedures for transformation of Aspergillus cells are described in EP238 023 and Yelton et al., 1984, Proceedings of the National Academy ofSciences USA 81: 1470-1474. A suitable method of transforming Fusariumspecies is described by Malardier et al., 1989, Gene 78: 147-156 or inWO 96/00787.

“Introduction” means introducing a vector comprising the nucleic acidsequence encoding the polypeptide into a filamentous fungal cell so thatthe vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector. Integration is generallyconsidered to be an advantage as the nucleic acid sequence is morelikely to be stably maintained in the cell. Integration of the vectorinto the chromosome occurs by homologous recombination, non-homologousrecombination, or transposition.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. These nucleic acid sequences may be anysequence that is homologous with a target sequence in the genome of thehost cell, and, furthermore, may be non-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook, et al., supra).

In a preferred embodiment, the filamentous fungal host cell is a cell ofa species of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma.

In a more preferred embodiment, the filamentous fungal cell is anAspergillus cell. In another more preferred embodiment, the filamentousfungal cell is an Acremonium cell. In another more preferred embodiment,the filamentous fungal cell is a Fusarium cell. In another morepreferred embodiment, the filamentous fungal cell is a Humicola cell. Inanother more preferred embodiment, the filamentous fungal cell is aMucor cell. In another more preferred embodiment, the filamentous fungalcell is a Myceliophthora cell. In another more preferred embodiment, thefilamentous fungal cell is a Neurospora cell. In another more preferredembodiment, the filamentous fungal cell is a Penicillium cell. Inanother more preferred embodiment, the filamentous fungal cell is aThielavia cell. In another more preferred embodiment, the filamentousfungal cell is a Tolypocladium cell. In another more preferredembodiment, the filamentous fungal cell is a Trichoderma cell.

In a most preferred embodiment, the filamentous fungal cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum cell. In an even most preferred embodiment, thefilamentous fungal cell is a Fusarium venenatum (Nirenberg sp. nov.). Inanother most preferred embodiment, the filamentous fungal cell is aHumicola insolens or Humicola lanuginosa cell. In another most preferredembodiment, the filamentous fungal cell is a Mucor miehei cell. Inanother most preferred embodiment, the filamentous fungal cell is aMyceliophthora thermophilum cell. In another most preferred embodiment,the filamentous fungal cell is a Neurospora crassa cell. In another mostpreferred embodiment, the filamentous fungal cell is a Penicilliumpurpurogenum cell. In another most preferred embodiment, the filamentousfungal cell is a Thielavia terrestris cell. In another most preferredembodiment, the Trichoderma cell is a Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichodermaviride cell.

Polypeptides having Transcriptional Activation Activity

Another aspect of the present invention relates to an isolatedpolypeptide selected from the group consisting of:

(a) a polypeptide which is encoded in a nucleic acid sequence whichhybridizes under low stringency conditions with (i) the nucleic acidsequence of SEQ ID NO: 1 or SEQ ID NO: 48; (ii) its complementarystrand, or (iii) a subsequence of SEQ ID NO: 1 or SEQ ID NO: 48 whichencodes a polypeptide fragment which has transcriptional activationactivity, wherein the low stringency conditions are defined byprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micro g/ml sheared and denatured salmon sperm DNA, and 25% formamide,and wash conditions are defined at 50° C. for 30 minutes in 2×SSC, 0.2%SDS;

(b) a polypeptide having an amino acid sequence which has at least 50%identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 49;

(c) an allelic variant of (a) or (b);

(d) a fragment of (a), (b), or (c), wherein the fragment hastranscriptional activation activity; and

(e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, oran allelic variant thereof.

The transcriptional activator may be isolated using techniques asdescribed herein. As defined herein, an “isolated” polypeptide is apolypeptide which is essentially free of other polypeptides, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

The present invention also relates to isolated polypeptides having anamino acid sequence which has a degree of identity to the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 49 of at least about 50%,preferably at least about 55%, preferably at least about 60%, preferablyat least about 65%, preferably at least about 70%, preferably at leastabout 75%, preferably at least about 80%, more preferably at least about85%, even more preferably at least about 90%, most preferably at leastabout 95%, and even most preferably at least about 97%, even morepreferred at least 99%, which have transcriptional activation activity.

In more preferred embodiment, the transcriptional activator of thepresent invention comprises the amino acid sequence of SEQ ID NO: 2 orSEQ ID NO: 49 or a fragment thereof, wherein the fragment retainstranscriptional activation activity. In a most preferred embodiment, thepolypeptide has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:49. A fragment of SEQ ID NO: 2 or SEQID NO: 49 is a polypeptide havingone or more amino acids deleted from the amino and/or carboxy terminusof this amino acid sequence. Preferably, a fragment of SEQ ID NO: 2 orSEQ ID NO: 49 contains at least the polypeptide sequence shown in SEQ IDNO: 3.

The amino acid sequences of the homologous polypeptides may differ fromthe amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 49 or SEQ ID NO: 3by an insertion or deletion of one or more amino acid residues and/orthe substitution of one or more amino acid residues by different aminoacid residues. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions that do not significantlyaffect the folding and/or activity of the protein; small deletions,typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (such as arginine, lysine and histidine), acidic amino acids(such as glutamic acid and aspartic acid), polar amino acids (such asglutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine and valine), aromatic amino acids (such as phenylalanine,tryptophan and tyrosine), and small amino acids (such as glycine,alanine, serine, threonine and methionine). Amino acid substitutions,which do not generally alter the specific activity are known in the artand are described, for example, by H. Neurath and R. L. Hill (1979. TheProteins, Academic Press, New York). The most commonly occurringexchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.

In a more preferred embodiment, a transcriptional activator of thepresent invention is obtained from an Aspergillus niger strain, morepreferably from Aspergillus niger AB4.1 (van Haringsveldt, W., et al.,1987. Mol. Gen. Genet. 206:71-75), and most preferably from Aspergillusniger 13PAP2, which has been deposited at DSM as DSM 12298, or a mutantstrain thereof, harbouring, e.g., the polypeptide with the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 49 or SEQ ID NO: 3.

In another preferred embodiment, the transcriptional activator of thepresent invention is the polypeptide encoded in the nucleic acidsequence contained in plasmid pEES, which is contained in Escherichiacoli DSM 12294 or the nucleic acid sequence shown in SEQ ID NO: 48encoding the polypeptide shown in SEQ ID NO: 49.

The present invention further relates to methods for producing thetranscriptional activator of the present invention comprising (a)cultivating a host cell harbouring a nucleic acid construct or anexpression vector comprising a nucleic acid sequence encoding thetranscriptional activator of the invention under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.

Host Cells having Altered Transcriptional Activation Activity

Another aspect of the present invention relates to a host cell which isa mutant of a parent fungal cell useful for the production of apolypeptide in which the parent cell comprises one or more nucleic acidsequences encoding a protease, the transcription of which is activatedby a transcriptional activator of the present invention, and the mutantcell produces less of the transcriptional activator and the protease(s)than the parent cell when cultured under the same conditions.

The mutant cell may be constructed using methods well known in the art;for example, by one or more nucleotide insertions or deletions of thegene encoding the transcriptional activator.

In a preferred embodiment the mutant cell is obtained by modification orinactivation of a nucleic acid sequence present in the cell andnecessary for expression of the transcriptional activator.

In a more preferred embodiment, the nucleic acid sequence is selectedfrom the group consisting of: (a) a nucleic acid sequence having atleast 70% identity with the nucleic acid sequence of SEQ ID NO: 1 or SEQID NO: 48; (b) a nucleic acid sequence encoding a polypeptide having anamino acid sequence which has at least 50% identity with the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 49; (c) a nucleic acid sequencewhich hybridizes under low stringency conditions with (i) the nucleicacid sequence of SEQ ID NO: 1 or SEQ ID NO: 48, or (ii) itscomplementary strand, (d) an allelic variant of (a), (b), or (c); (e) asubsequence of (a), (b), (c), or (d), wherein the subsequence encodes apolypeptide fragment which has transcriptional activation activity; and(f) a subsequence of (a), (b) (c), or (d), wherein the subsequenceencodes a polypeptide with the amino acid sequence of SEQ ID NO: 3.

In another preferred embodiment the reduced expression of thetranscriptional activator in the mutant cell is obtained by modificationor inactivation of a control sequence required for the expression of thetranscriptional activator. The term “control sequence” is defined,supra, in the section entitled “Nucleic Acid Constructs.” In a morepreferred embodiment the control sequence in the mutant cell is apromoter sequence or a functional part thereof, i.e., a part, which issufficient for affecting expression of the nucleic acid sequence. Othercontrol sequences for possible modification include, but are not limitedto, a leader, a polyadenylation sequence, a propeptide sequence, asignal sequence, and a transcription terminator.

Modification or inactivation of the gene may be performed by subjectingthe parent cell to mutagenesis and selecting for mutant cells in whichthe capability to produce a transcriptional activator has been reduced.The mutagenesis, which may be specific or random, may be performed, forexample, by use of a suitable physical or chemical mutagenizing agent,by use of a suitable oligonucleotide, or by subjecting the DNA sequenceto PCR generated mutagenesis. Furthermore, the mutagenesis may beperformed by use of any combination of these mutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor mutant cells exhibiting reduced expression of the gene.

Modification or inactivation of the gene may be accomplished byintroduction, substitution, or removal of one or more nucleotides in thegene's nucleic acid sequence or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change of the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the fungal cell expressing the geneto be modified, it is preferred that the modification be performed invitro as exemplified below.

An example of a convenient way to inactivate or reduce expression of thegene by a fungal cell of choice is based on techniques of genereplacement or gene interruption. For example, in the gene interruptionmethod, a nucleic acid sequence corresponding to the endogenous gene orgene fragment of interest is mutagenized in vitro to produce a defectivenucleic acid sequence which is then transformed into the parent cell toproduce a defective gene. By homologous recombination, the defectivenucleic acid sequence replaces the endogenous gene or gene fragment. Itmay be desirable that the defective gene or gene fragment also encodes amarker, which may be used for selection of transformants in which thenucleic acid sequence has been modified or destroyed.

Alternatively, modification or inactivation of the gene may be performedby established anti-sense techniques using a nucleotide sequencecomplementary to the nucleic acid sequence of the gene. Morespecifically, expression of the gene by a filamentous fungal cell may bereduced or eliminated by introducing a nucleotide sequence complementaryto the nucleic acid sequence, which may be transcribed in the cell andis capable of hybridizing to the mRNA produced in the cell. Underconditions allowing the complementary anti-sense nucleotide sequence tohybridize to the mRNA, the amount of protein translated is thus reducedor eliminated.

A nucleic acid sequence complementary to the nucleic acid sequence ofSEQ ID NO: 1 or SEQ ID NO: 48 may be obtained from any microbial source.The preferred sources are fungal sources, e.g., yeast and filamentousfungi as described supra. Preferred filamentous fungal sources include,but are not limited to, species of Acremonium, Aspergillus, Fusarium,Humicola, Myceliophthora, Mucor, Neurospora, Penicillium, Phanerochaete,Thielavia, Tolypocladium, and Trichoderma. Preferred yeast sourcesinclude, but are not limited to, species of Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia.Furthermore, the nucleic acid sequence may be native to the filamentousfungal cell.

In another preferred embodiment, the parent cell harbours a gene havinga nucleic acid sequence encoding a polypeptide with an amino acidsequence which has at least 50% identity with the amino acid sequence ofSEQ ID NO: 2 or SEQID NO: 49.

In another preferred embodiment, the parent cell harbours a gene havinga nucleic acid sequence with at least 70% identity with the nucleic acidsequence of SEQ ID NO: 1 or SEQ ID NO: 48.

In another preferred embodiment, the mutant cell harbours a nucleic acidsequence, which has been modified or inactivated by any of the methodsdescribed above and produces less of a protease or a combination ofproteases than the parent cell when cultured under identical conditions.The mutant cell produces preferably at least about 25% less, morepreferably at least about 50% less, even more preferably at least about75% less, and even more preferably at least about 95% less of a proteaseor a combination of proteases than the parent cell when cultured underidentical conditions.

In an even more preferred embodiment, the mutant cell producesessentially undetectable amounts of a protease or combination ofproteases than the parent cell when cultured under identical conditions.

The protease(s) may be assayed using known methods. In one such method,an aliquot of a 48 hour culture media is incubated with ³H-labelledsperm whale myoglobin at pH 4.0 and the radioactivity in the TCA-solublefraction is measured (van Noort, J. M., et al., 1991. Anal. Biochem198:385-390). Other methods have been described for identifying, e.g.,aspartic proteinase A. of A. niger (Takahashi, K., 1991. Meth. inEnzymol. 248:146-155), endopeptidases (Morihara, K., 1995. Meth. inEnzymol. 248:242-253), carboxypeptidases (Reminton, J., and Breddam, K.,1994. Meth. in Enzymol. 244:231-248), dipeptidyl peptidase (Ikehara, Y.,et al., 244:215-227), and aminopeptidases (Little, G., et al., 1976.Meth. in Enzymol. 45:495-503).

In another preferred embodiment, the mutant cell harbours at least onecopy of a nucleic acid sequence encoding a polypeptide of interest.

Another aspect of the present invention relates to a host cell usefulfor the production of a polypeptide wherein the host cell is a mutant ofa parent fungal cell in which the mutant (a) produces more of thetranscriptional activator of the present invention as compared to theparent cell when cultured under the same conditions; and (b) comprises aDNA sequence encoding the polypeptide, the transcription of which isactivated by the transcriptional activator.

In a preferred embodiment, the host cell produces more of thetranscriptional activator than the parent cell when cultured under thesame conditions by introducing into the parent cell one or more copiesof (i) a nucleic acid sequence encoding a transcriptional activator,(ii) a nucleic acid construct comprising a nucleic acid sequenceencoding a transcriptional activator, or (iii) an expression vector asdefined above in the section “Expression Vectors”.

The nucleic acid construct comprising a nucleic acid sequence encoding atranscriptional activator of the present invention may also comprise oneor more nucleic acid sequences which encode one or more factors that areadvantageous for directing the expression of the polypeptide, e.g., atranscriptional activator (e.g., a trans-acting factor), a chaperone,and a processing protease. Any factor that is functional in thefilamentous fungal cell of choice may be used in the present invention.The nucleic acids encoding one or more of these factors are notnecessarily in tandem with the nucleic acid sequence encoding thepolypeptide.

An activator is a protein, which activates transcription of a nucleicacid sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal9: 1355-1364; Jarai and Buxton, 1994, Current Genetics 26: 2238-244;Verdier, 1990, Yeast 6: 271-297). The nucleic acid sequence encoding anactivator may be obtained from the genes encoding Saccharomycescerevisiae heme activator protein 1 (hap1), Saccharomyces cerevisiaegalactose metabolizing protein 4 (gal4), Aspergillus nidulans ammoniaregulation protein (areA), and Aspergillus oryzae alpha-amylaseactivator (amyR). For further examples, see Verdier, 1990, supra andMacKenzie et al., 1993, Journal of General Microbiology 139: 2295-2307.

A chaperone is a protein which assists another polypeptide in foldingproperly (Hartl et al., 1994, TIBS 19: 20-25; Bergeron et al., 1994,TIBS 19: 124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189; Craig, 1993, Science 260: 1902-1903; Gething and Sambrook,1992, Nature 355: 33-45; Puig and Gilbert, 1994, Journal of BiologicalChemistry 269: 7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; Robinson et al., 1994, Bio/Technology 1: 381-384; Jacobs etal., 1993, Molecular Microbiology 8: 957-966). The nucleic acid sequenceencoding a chaperone may be obtained from the genes encoding Aspergillusoryzae protein disulphide isomerase or Saccharomyces cerevisiaecalnexin, Saccharomyces cerevisiae BiP/GRP78, and Saccharomycescerevisiae Hsp70. For further examples, see Gething and Sambrook, 1992,supra, and Hartl et al., 1994, supra.

A processing protease is a protease that cleaves a propeptide togenerate a mature biochemically active polypeptide (Enderlin andOgrydziak, 1994, Yeast 10: 67-79; Fuller et al., 1989, Proceedings ofthe National Academy of Sciences USA 86: 1434-1438; Julius et al., 1984,Cell 37: 1075-1089; Julius et al., 1983, Cell 32: 839-852; U.S. Pat. No.5,702,934). The nucleic acid sequence encoding a processing protease maybe obtained from the genes encoding Saccharomyces cerevisiaedipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, Yarrowialipolytica dibasic processing endoprotease (xpr6), and Fusariumoxysporum metalloprotease (p45 gene).

In a more preferred embodiment, the nucleic acid sequence encoding thetranscriptional activator is operably linked to a promoter, or afunctional part thereof, which is stronger than the correspondingpromoter of the parent cell. In an even more preferred embodiment, thepromoter, or a functional part thereof, mediates the expression of agene encoding an extracellular protease, such as the Aspergillus oryzaealkaline protease, A. oryzae neutral metalloprotease, A. nigeraspergillopepsin protease, Fusarium oxysporum trypsin-like protease orF. venenatum trypsin.

The present invention also relates to a host cell useful for theproduction of a polypeptide wherein the host cell is a mutant of aparent fungal cell in which the mutant comprises

a) a modification or inactivation of a transcriptional activator of thepresent invention, or a regulatory sequence thereof, and

b) (i) an inducible promoter operably linked to a nucleic acid sequenceencoding a transcriptional activator of the present invention, and (ii)a promoter sequence to which the transcriptional activator can bind,operably linked to a nucleic acid sequence encoding the polypeptide,wherein (i) and (ii) can be introduced simultaneously or sequentially.

The inactive form of the transcriptional activator in (a) above isobtained by inactivation or modification of a nucleic acid sequencepresent in the cell and necessary for the expression of the nativetranscriptional activator according to any of the methods as disclosedsupra. In a preferred embodiment the inactivation or modification isobtained by methods, which include, but are not limited to, one or morenucleotide insertions, deletions or substitutions, specific or randommutagenesis, gene replacement or gene interruption, and anti-sensetechniques using a nucleotide sequence complementary to the nucleic acidsequence of the transcriptional activator. In another preferredembodiment, the inactive form of the native transcriptional activator isobtained by inactivation or modification of a control sequence requiredfor the expression of the transcriptional activator.

In another preferred embodiment, the nucleic acid sequence encoding thenative transcriptional activator has the sequence set forth in SEQ IDNO: 1 or SEQ ID NO: 48. In another preferred embodiment, thetranscriptional activator comprises the polypeptide having the aminoacid sequence in SEQ ID NO: 3.

The inducible promoter sequence in (b) above may be any promotersequence, or a functional part thereof, wherein the transcriptioninitiation activity of the promoter can be induced according to thefermentation conditions. Preferably, the induction is mediated by acarbon or nitrogen catabolite. In a preferred embodiment, the promoteris the amdS promoter of Aspergillus nidulans or A. oryzae, the niaDpromoter of A. nidulans, A. oryzae or A. niger, the niiA promoter ofAspergillus species, the alkaline phosphatase promoter of Aspergillussp., the acid phosphatase promoter of Aspergillus sp., or the alcApromoter of A. niger.

In another preferred embodiment, the host cell further comprises apromoter sequence, wherein the promoter sequence can be activated by thetranscriptional activator and is operably linked to the nucleic acidsequence encoding the polypeptide.

The promoter sequence activated by the transcriptional activator of thepresent invention may be any promoter sequence, or a functional partthereof, selected from the group which includes but is not limited topromoters obtained from the genes encoding Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, theNA2-tpi promoter (a hybrid of the promoters from the genes encodingAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Particularly preferred promoters for use in filamentous fungalcells are a promoter, or a functional part thereof, from a proteasegene; e.g., from the Fusarium oxysporum trypsin-like protease gene (U.S.Pat. No. 4,288,627), Aspegillus oryzae alkaline protease gene (alp),Aspergillus niger pacA gene, Aspergillus oryzae alkaline protease gene,A. oryzae neutral metalloprotease gene, A. niger aspergillopepsinprotease gene, or F. venenatum trypsin gene.

In another preferred embodiment, the host cell harbours at least onecopy of a nucleic acid sequence encoding a polypeptide.

In another preferred embodiment, the host cell, which expresses thetranscriptional activator of the present invention produces less of oneor more native proteases than the parent cell when cultured underidentical conditions. The protease(s) may be assayed using any of themethods described above. In a more preferred embodiment, an aliquot froma 48-hour culture media is incubated with ³H-labelled sperm whalemyoglobin at pH 4.0 and the radioactivity in the TCA-soluble fraction ismeasured (van Noort, J. M., et al., supra).

The nucleic acid constructs described herein may be introduced into aparent fungal cell according to any of the methods as described supra inthe section, “Host Cells” to obtain a host cell useful for theproduction of a polypeptide. In a preferred embodiment the nucleic acidconstruct is integrated into the chromosome of the cell. In anotherpreferred embodiment the nucleic acid construct is maintained as aself-replicating extra-chromosomal vector.

It will be understood that the methods of the present invention are notlimited to a particular order for obtaining the mutant fungal cell. Themodification of the second nucleic acid sequence may be introduced intothe parent cell at any step in the construction of the cell for theproduction of a polypeptide.

Producing a Polypeptide

Another aspect of the present invention relates to methods of producinga polypeptide in a host cell of the present invention, comprising: (a)cultivating the host cell which harbours a gene encoding the polypeptidein a nutrient medium suitable for production of the polypeptide; and (b)recovering the polypeptide from the nutrient medium of the host cell.

In one embodiment, the host cell which is a mutant of a parent fungalcell in which the parent cell comprises one or more nucleic acidsequences encoding a protease, the transcription of which is activatedby a transcriptional activator of the present invention, and the mutantcell produces less of the transcriptional activator and the protease(s)than the parent cell when cultured under the same conditions.

In another embodiment, the host cell is a mutant of a parent fungal cellin which the mutant (a) produces more of the transcriptional activatorof the present invention as compared to the parent cell when culturedunder the same conditions; and (b) comprises a DNA sequence encoding thepolypeptide, the transcription of which is activated by thetranscriptional activator.

In another embodiment, the host cell is a mutant of a parent fungal cellin which the mutant comprises (a) a modification or inactivation of atranscriptional activator of the present invention or a regulatorysequence thereof, and (b) an inducible promoter operably linked to anucleic acid sequence encoding a transcriptional activator of thepresent invention and a promoter sequence to which the transcriptionalactivator can bind, operably linked to a nucleic acid sequence encodingthe polypeptide, wherein (i) and (ii) can be introduced simultaneouslyor sequentially.

The host cells of the present invention are cultivated in a nutrientmedium suitable for production of the polypeptide of interest usingmethods known in the art. For example, the cells may be cultivated byshake flask cultivation, small-scale or large-scale fermentation(including continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermentors performed in a suitable mediumand under conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art (see, e.g., Bennett, J. W. and LaSure, L.,eds., More Gene Manipulations in Fungi, Academic Press, CA, 1991).Suitable media are available from commercial suppliers or may beprepared using published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the polypeptide is secreted intothe nutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it is recovered from celllysates.

The resulting polypeptide may be isolated by methods known in the art.For example, the polypeptide may be isolated from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray drying, evaporation, or precipitation. Theisolated polypeptide may then be further purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing, differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

The polypeptide may be detected using methods known in the art that arespecific for the polypeptide. These detection methods may include use ofspecific antibodies, formation of an enzyme product, disappearance of anenzyme substrate, or SDS-PAGE. For example, an enzyme assay may be usedto determine the activity of the polypeptide. Procedures for determiningenzyme activity are known in the art for many enzymes.

In the methods of the present invention, the host cell produces at leastabout 20% more, preferably at least about 50% more, more preferably atleast about 100% more, even more preferably at least about 200% more,and most preferably at least about 300% more of the polypeptide than acorresponding parent cell when cultivated under the same conditions.

The polypeptide may be any polypeptide whether native or heterologous tothe mutant filamentous fungal cell. The term “heterologous polypeptide”is defined herein as a polypeptide, which is not produced by a cell. Theterm “polypeptide” is not meant herein to refer to a specific length ofthe encoded produce and therefore encompasses peptides, oligopeptidesand proteins. The polypeptide may also be a recombinant polypeptide,which is a polypeptide native to a cell, which is encoded by a nucleicacid sequence, which comprises one or more control sequences foreign tothe nucleic acid sequence, which are involved in the production of thepolypeptide. The polypeptide may be a wild-type polypeptide or a variantthereof. The polypeptide may also be a hybrid polypeptide, whichcontains a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides where one or more ofthe polypeptides may be heterologous to the cell. Polypeptides furtherinclude naturally occurring allelic and engineered variations of theabove-mentioned polypeptides.

In a preferred embodiment, the polypeptide is an antibody or portionsthereof, an antigen, a clotting factor, an enzyme, a hormone or ahormone variant, a receptor or portions thereof, a regulatory protein, astructural protein, a reporter, or a transport protein.

In a more preferred embodiment, the enzyme is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase.

In an even more preferred embodiment, the enzyme is an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, deoxyribonuclease, dextranase, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

In another even more preferred embodiment, the polypeptide is humaninsulin or an analog thereof, human growth hormone, erythropoietin, orinsulinotropin.

The nucleic acid sequence encoding a heterologous polypeptide may beobtained from any prokaryotic, eukaryotic, or other source. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide isproduced by the source or by a cell in which a gene from the source hasbeen inserted.

In the methods of the present invention, the mutant filamentous fungalcells may also be used for the recombinant production of polypeptides,which are native to the cell. The native polypeptides may berecombinantly produced by, e.g., placing a gene encoding the polypeptideunder the control of a different promoter to enhance expression of thepolypeptide, to expedite export of a native polypeptide of interestoutside the cell by use of a signal sequence, and to increase the copynumber of a gene encoding the polypeptide normally produced by the cell.The present invention also encompasses, within the scope of the term“heterologous polypeptide”, such recombinant production of polypeptidesnative to the cell, to the extent that such expression involves the useof genetic elements not native to the cell, or use of native elementswhich have been manipulated to function in a manner that do not normallyoccur in the filamentous fungal cell. The techniques used to isolate orclone a nucleic acid sequence encoding a heterologous polypeptide areknown in the art and include isolation from genomic DNA, preparationfrom cDNA, or a combination thereof. The cloning of the nucleic acidsequences from such genomic DNA can be effected, e.g., by using the wellknown polymerase chain reaction (PCR). See, for example, Innis et al.,1990, PCR Protocols: A Guide to Methods and Application, Academic Press,New York. The cloning procedures may involve excision and isolation of adesired nucleic acid fragment comprising the nucleic acid sequenceencoding the polypeptide, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into the mutantfungal cell where multiple copies or clones of the nucleic acid sequencewill be replicated. The nucleic acid sequence may be of genomic, cDNA,RNA, semisynthetic, synthetic origin, or any combinations thereof.

In the methods of the present invention, heterologous polypeptides mayalso include fused or hybrid polypeptides in which another polypeptideis fused at the N-terminus or the C-terminus of the polypeptide orfragment thereof. A fused polypeptide is produced by fusing a nucleicacid sequence (or a portion thereof) encoding one polypeptide to anucleic acid sequence (or a portion thereof) encoding anotherpolypeptide. Techniques for producing fusion polypeptides are known inthe art, and include, ligating the coding sequences encoding thepolypeptides so that they are in frame and expression of the fusedpolypeptide is under control of the same promoter(s) and terminator. Thehybrid polypeptides may comprise a combination of partial or completepolypeptide sequences obtained from at least two different polypeptideswherein one or more may be heterologous to the mutant fungal cell. Anisolated nucleic acid sequence encoding a heterologous polypeptide ofinterest may be manipulated in a variety of ways to provide forexpression of the polypeptide. Expression will be understood to includeany step involved in the production of the polypeptide including, butnot limited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.Manipulation of the nucleic acid sequence encoding a polypeptide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying nucleic acidsequences utilizing cloning methods are well known in the art.

The present invention is further described by the following examples,which should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

AB4.1: a strain of Aspergillus niger which is a cspA1 pyrG1 derivativeof strain ATTC 9029 (van Hartingsveldt, W., et al., 1987. Mol. Gen.Genet. 206:71-75; Bos, C. J., et al., Curr. Genet. 14:437-443)

AB1.13: a protease deficient strain of Aspergillus niger derived from UVmutagenesis of AB4.1 (Mattern, I. E., et al., 1992. Mol. Gen. Genet.234:332-336)

13PAP2: an AB1.13 derivative containing multiple copies of the A.nidulans amdS gene (Corrick, R. A., et al, 1987. Gene 53: 63-71) undercontrol of the pepA promoter of A. niger (Jarai G. and Buxton F. 1994.Curr Genet 26:238-244). The strain has a protease deficient phenotypeand is unable to grow on medium containing acetamide as the solenitrogen source. Strain 13PAP2 has been deposited at DSM under the nameDSM No. 12298.

4PAP6: an AB4.1 derivative containing multiple copies of the of A.nidulans amdS gene under control of the pepA promoter of A. niger. Thestrain does not have a protease deficient phenotype and is able to growon medium containing acetamide as the sole nitrogen source.

N402: a strain of Aspergillus niger, deposited at the ATCC (ManassasVa., USA) as ATCC Number: 64974

MC1046: a strain of E. coli, deposited at the ATCC as ATCC Number: 35467

A.oryzae IFO4177: available from Institute for Fermentation, Osaka;17-25 Juso Hammachi 2-Chome Yodogawa-Ku, Osaka, Japan.

HowB101: described in WO 97/35956.

Plasmids

pPAP: constructed as described below in Example 1 and shown in FIG. 1

pAopyrGcosArp1: constructed as described below in Example 1 and shown inFIG. 2

pEES1: constructed as described below in Example 1 and shown in FIG. 3

p3SR2: contains the A. nidulans amdS gene as described by C. M. Corrick,A. P. Twomey, and M. J. Hynes (1987. Gene 53: 63-71)

pABPYRG*-Not: contains an inactivated pyrG gene as described by Verdoes,J. C., et al. (1994. Gene 145: 179-187)

pHelp1: contains the pyrG gene from A. oryzae as a selective marker andthe AMA1 sequences which enable autonomous replication in A. niger,cloned into the E. coli vector pIC20R, as described by Gems, D., et al.(1991. Gene 98: 61-67)

pAnscos1: contains two cos sites as described by Osiewacz, H. D. (1994.Curr. Genet. 26: 87-90)

pAO4-2: contains the A. oryzae pyrG gene as described by DeRuiter-Jacobs, Y. M. J. T., et al. (1989. Curr. Genet. 16: 159-163)

pAO4-13: contains the A. oryzae pyrG gene as described by DeRuiter-Jacobs, Y. M. J. T., et al. (1989. Curr. Genet. 16:159-163)

pUC19: as described by Yanisch-Perron, C., Vieira, J. and Messig, J.(1985, Gene 33:103-119)

pDV8: described in Example 8 and shown in FIG. 7.

pJaL554: described in Example 8 and shown in FIG. 8

Deposit of Biological Materials

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Deutsche Sammlung von Microorganismen undZellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany andgiven the following accession number:

Deposit Accession Number Date of Deposit Escherichia coil, pEES DSM12294 1998-07-14 Aspergilius niger 13PAP2 DSM 12298 1998-07-14

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Example 1

Cloning of the A. niger prtT Transcriptional Activator

The prtT gene was cloned from 13PAP2, an A. niger mutant strain which isunable to express the amdS gene regulated by the pepA protease genepromoter and has a protease deficient phenotype (prt⁻).

Construction of the A. niger 13PAP2 Reporter Strain

The plasmid pPA1 was contructed by ligation of the following threefragments:

1) the E. coli vector pBlueScript II SK (Stratagene Cloning Systems, LaJolla Calif., USA) digested with EcoRI and KpnI;

2) a 1.4 kb EcoRI/BamHI restriction fragment containing the 1.2 kbpromoter region of the pepA gene linked to about 130 bp of the amdScoding sequence from the start codon to an internal BamHI site,amplified by PCR; and

3) a 2.1 kb BamHI/KpnI fragment from p3SR2 which contains most of the A.nidulans amdS gene

Fragment 2 was constructed in two steps. In a first step genomic DNAfrom A. niger N402 prepared from protoplasts as described below in thesection “Construction of the Cosmid Library” was used as the template,and the two oligonucleotides shown below, pepApr and pepA/amdS, wereused as primers:

PepApr: (SEQ ID NO: 6) CGG AAT TCG CAT GCT GGA GGT GCT TCT AA pepA/amdS:(SEQ ID NO: 7) TTC CCA GGA TTG AGG CAT TTT GAC CAC GAG AAT

The 1200 bp PCR product obtained from this reaction was then used as aprimer in a second PCR reaction together with the oligonucleotideMBL1213 shown below, and plasmid p3SR2 as the template. MBL1213: TAA CTTCCA CCG AGG TC (SEQ ID NO: 8)

The product obtained by ligation of the three fragments described abovewas subsequently transfected into E. coli DH5α.

In the final construction procedure, pPA1 was digested with NotI andligated to a 3.8 kb NotI fragment from pABPYRG*-Not, resulting inplasmid pPAP which is shown in FIG. 1. pPAP was transformed into A.niger AB 1.13, and a transformant with pPAP integrated into the pyrGlocus in multicopy was isolated. A spontaneous 5 fluorotic acid (FOA)resistant, uridine-requiring mutant of this transformant that could becomplemented with the pyrG gene was named 13PAP2.

Construction of pAopyrGcosArp1

The plasmid pAopyrGcosArp1 was constructed by ligation and subsequenttransfection into E. coli DH5□ of the following three fragments:

1) the E. coli vector pHelp1 cut with Acc65I and BamHI

2) a 3.0 kb BamHI/HindIII fragment from pAnscos1 containing two cossites

3) a 3.2 kb Acc65I/HindIII fragment from pAO4-2 containing the A. oryzaepyrG gene

The resulting plasmid, pAopyrGcosArp1, is self-replicating in Aspergilliand can be selected for by growth on medium lacking uridine.pAopyrGcosArp1 is depicted in FIG. 2.

Construction of the Cosmid Library.

A cosmid library of Aspergillus niger was constructed using the“SuperCos1 cosmid vector kit” (Stratagene Cloning Systems, La JollaCalif., USA) according to the supplier's instructions.

Genomic DNA from A. niger N402 was prepared from protoplasts made bystandard procedures.

After isolation the protoplasts were pelleted by centrifugation at 2000rpm for 10 minutes in a Beckman GS-6R; the pellet was then suspended ina buffer containing 22.5 mM tri-isonaphtalene sulphonic acid, 275 mMpara-aminosalicylic acid, 0.2 M Tris-HCl (pH 8.5), 0.25 M NaCl and 50 mMEDTA immediately followed by addition of 1 volume of phenol/chloroform(1:1). After careful mixing and centrifugation at 3000 rpm for 20minutes the aqueous phase was decanted and DNA was precipitated usingstandard procedures.

The size of the genomic DNA was analysed by electrophoresis on a 0.3%agarose gel run for 20 hours at 30 volts at 4° C. The ethidium bromidestained gel showed that the recovered DNA ranged in size from 50 togreater than 100 kb. The DNA was partially digested using MboI. The sizeof the digested DNA was 30 to 50 kb as determined by the same type ofgel analysis as above. The pAopyrGcosArp1 vector, purified using a kitfrom QIAGEN (Venlo, The Netherlands) following the manufacturer'sinstructions, was digested with BamHI, dephosphorylated and gelpurified. Ligation and packaging were performed following standardprocedures.

After titration of the library, all of the packaging mix from a singleligation and packaging was transfected into the host cell, MC1046, andplated on 50 μg/ml ampicillin LB plates. Approximately 40,000 colonieswere obtained. Cosmid preparations from 10 colonies showed that they allhad inserts of the expected size. The 40,000 colonies were then soakedin LB medium and scraped off of the plates, then aliquoted for storagein 15% glycerol at −80° C. This represents an approximate 40-foldamplification of the A. niger genome.

Selection of A. niger PrtT Clones

Cosmid DNA was prepared from the library and introduced into 13PAP2according to the transformation procedure described by P. J. Punt and C.A. M. J. J. Van Den Hondel (1992. Methods Enzymol 216: 447-457).Repeated efforts to select for the pyrG marker only resulted in arecovery of between 4000 to 30,000 transformants. A double selection forthe pyrG marker and growth on medium containing acetamide as the solenitrogen source resulted in a total of 65 primary transformants fromfive different experiments.

Each primary transformant was screened for protease activity, growth onmedium containing acetamide as the sole nitrogen source and instabilityof these two characteristics. An acetamidase⁺ phenotype, screened bygrowth on medium containing acetamide, is an indication of acetamidaseactivity resulting from activation of the pepA promoter in the reportercassette in which the pepA promoter is linked to the amdS codingsequence. A protease⁺ phenotype was screened using minimal medium platescontaining dialyzed skim milk as the sole nitrogen source (Mattern, I.E., et al., 1992. Mol Gen Genet 234:332-336). On these plates thewild-type AB4.1 strain makes a clear halo whereas the AB1.13 mutantproduces a very small halo. This difference is not due to differences inthe activity of pepA since a pepA deleted strain can also produce alarge halo on these plates. Therefore, a large halo on milk platesindicates activation of other extracellular proteases.

Instability was tested by growing diluted spore stocks on mediumcontaining uridine. Single-spore-derived colonies were picked from theseplates and tested for protease activity and growth on acetamide. Thescreening results revealed that in more than 70% of the colonies bothcharacteristics were lost. Therefore, the two phenotypes were eitherlost or retained together, indicating that activation of the pepApromoter and other protease promoters is coordinately regulated andlinked to the presence of the pyrG marker. The gene responsible for thisphenotype was named prtT. Twelve acetamidase⁺, protease⁺ transformantswere then isolated.

Isolation of the A.niger PrtT Gene

In order to rescue the prtT gene from the acetamidase⁺, protease⁺transformants of 13PAP2, DNA was prepared from mycelium grown in minimalmedium as previously described. This DNA was used in an attempt totransform competent E. coli DH5□ cells. Several hundreds ofampicillin-resistant colonies were obtained. DNA analysis showed theyall contained sequences derived from the pHelp1 plasmid. Cosmid DNAisolated from E. coli colonies was then retransformed into 13PAP2. TwoDNA samples gave rise to transformants, which showed both growths onacetamide containing medium and increased protease activity. DNA fromone of the cosmids, ACR1, was then digested with several restrictionenzymes. The resulting fragments were then co-transformed withpAopyrGcosArp1 into strain 13PAP2. EcoRI, PstI, BamHI and KpnI digestionof ACR1 gave rise to transformants capable of growth on acetamide andhigh protease activity, whereas SalI and HindIII digests did not.Because EcoRI digestion gave the simplest pattern, separate EcoRIfragments were gel-isolated and with pAopyrGcosArp1 used to cotransform13PAP2. Only one fragment, a 15 kb EcoRI fragment, gave rise totransformants capable of growth on acetamide-containing medium. Thisfragment was subcloned in pBluescript II SK in order to subclone prtTfrom the cosmid. Since the insert of this clone was still rather large,separate PstI bands were gel isolated and each was co-transformed withpAopyrGcosArp1 into 13PAP2. Only one band, a 2.5 kb PstI fragment, gaverise to transformants that could grow on acetamide-containing medium.This fragment was subcloned in pBlueScript II SK. Four subclones, ClE0.7, ClE 1.8, NcE 1.1 and NcE 1.4, were constructed from this plasmidbased on the restriction map. In addition, a 6.5 kb SstI/EcoRI fragmentencompassing the 2.5 kb PstI fragment was subcloned, resulting in pEES1(shown in FIG. 3).

Southern blot analysis of genomic DNA from AB4.1 showed the presence ofonly one copy of prtT.

Example 2

Sequencing of the A. niger PrtT Gene and Analysis of the Sequence

All sequence reactions were prepared using dRhodamine Terminator CycleSequencing Kits or BigDye™ Terminator Cycle Sequencing Kits from thePerkin-Elmer Corporation (Branchburg N.J., USA). The reactions were runon an ABI PRISM® 377 DNA Sequencer (Perkin-Elmer Corporation) followingthe manufacturer's instructions.

The prtT gene was sequenced from the genomic clones ClE 0.7, ClE 1.8,NcE 1.1, NcE 1.4 and pEES1. The sequence specific primers used arelisted below:

(SEQ ID NO: 9) 122958: CGA TCG ATG ACT GCC TGT (SEQ ID NO: 10) 122956:AGA GAC ACA TAG TGC CTT (SEQ ID NO: 11) 122959: GCT TAT AGT CGA TAG CGC(SEQ ID NO: 12) 122960: CCT CTC TCC AGC GAT GGT (SEQ ID NO: 13) 122962:ATG GAA TAC ATA CTG CTT (SEQ ID NO: 14) 122961: ATG AAA CCC ACT GTA GCT(SEQ ID NO: 15) 122963: TGC TCG ATA AGC GGG TCC (SEQ ID NO: 16) 122964:AAT CTT ATG GAC CCG CTT (SEQ ID NO: 17) 124289: CCC CGG GAA ACA AGA ACAGG (SEQ ID NO: 18) 124290: GTT GGC GGA CCT TGA CTA TG (SEQ ID NO: 19)125112: ACA GCT ACA GTG GGT TTC ATC T (SEQ ID NO: 20) 125111: AGT CAACGG GGG AAG TCT C (SEQ ID NO: 21) 128330: CTA GCA GCG TAT CGG TCA GC(SEQ ID NO: 22) 130887: CTT GGA AAA GAA ACG ATA G (SEQ ID NO: 23)130888: AAC GTA CGC TTT CCT CCT T (SEQ ID NO: 24) 134135: GGG TCC GTCCAG TCC GTT CTT (SEQ ID NO: 25) −48 reverse: AGC GGA TAA CAA TTT CAC ACAGGA (SEQ ID NO: 26) −40 universal: GTT TTC CCA GTC ACG AC

A mutant allele of the gene was obtained by PCR amplification of genomicDNA isolated from the mutant strain AB1.13 using the following primers:

PstI: TC ATC CCT GGT GTT ACT GC (SEQ ID NO: 27) PstII: C ATG GAT TGG CTGGCC G (SEQ ID NO: 28)

The complete DNA sequence of the prtT gene is shown in SEQ ID NO: 1. Thesequence of the PCR fragment of the mutant allele is shown in SEQ ID NO:4.

Analysing the DNA sequence SEQ ID NO: 1 using the computer softwareNetgene 2 (S. M. Hebsgaard, P. G. Korning, N. Tolstrup, J. Engelbrecht,P. Rouze, S. Brunak (1996. Nucleic Acids Research 24: 3439-3452)suggested the existence of 5 exons (see annotations to SEQ ID NO 1).

Analysis of the A. niger PrtT cDNA

mRNA was purified from total RNA (isolated according to the DNAisolation method described above in Example 1) using a commercialpoly(A)⁺ RNA isolation kit (Pharmacia, Uppsala SE) from a culture of A.niger grown under conditions favourable for protease production (J. P.T. W. Van Den Hombergh, et al., 1997. Eur. J. Biochem. 247:605-613).Double stranded cDNA was prepared using standard procedures and used forPCR reactions with the following primers:

oligo-dT primer: T₂₀N

Prt270n: TACTCTCCAGATTGCCTG (SEQ ID NO: 29) Prt1420r: TGAGATACCACTCAGCAG(SEQ ID NO: 30) prt1350n: TGCACTTCTCTGTCTCTG (SEQ ID NO: 31) Prt2365r:GACTTCTGGCATCAGTTG (SEQ ID NO: 32) prt2320n: CTCATGGATGGCATGATC (SEQ IDNO: 33)

A PCR reaction with the primers Prt270n and Prt1420r produced a fragmentof approximately 1.0 kb. The fragment was cloned into a pGEM-T vector(Promega Corp., Madison Wis., USA), and the insert in the resultingplasmid was sequenced using the primers 122958, 122960, −40 universaland −48 reverse. The result confirmed the presence of two introns inthis part of the gene.

A second PCR reaction with the primers Prt1350n and Prt2365r produced afragment of approximately 0.9 kb. This fragment was also cloned in apGEM-T vector, and the insert in the resulting plasmid was sequencedusing the primers 124289, 124290, −40 universal and −48 reverse. Theresult confirmed the presence of a single intron in this part of thegene.

Another PCR reaction with the oligo-dT primer and primer Prt2320nproduced a fragment of approximately 350 bp. This fragment was alsocloned in a pGEM-T vector. Sequencing of the insert using primers −40universal and −48 reverse showed that the fragment contained the 3′ partof prtT and confirmed the presence of another intron.

The deduced protein sequence of the translated prtT gene is shown in SEQID NO: 2. The deduced protein sequence of the translated mutant alleleprt13 is shown in SEQ ID NO: 5. A comparison of SEQ ID NO: 2 and SEQ IDNO: 5 indicates that the only difference between the two is in position112 where the leucine residue in the translated prtT gene is replaced byproline in the translated prt13 gene.

Analysis of the deduced PrtT protein sequence reveals the presence of aZinc(II)2Cys6 binuclear cluster DNA binding motif (SEQ ID NO: 2,residues 47-81). This motif defines the GAL4 class of fungaltranscriptional activators (Reece, M. J., and Ptashne, M. 1993. Science261: 909-911). The presence of the motif in the prtT gene stronglyindicates that prtT is a transcriptional activator.

Example 3

Disruption of the PrtT Gene in a Wild-Type A. niger Strain

A plasmid was constructed in which the upstream and downstream sequencesof the prtT gene are separated by the A. oryzae pyrG gene. Plasmid pEES1was digested with MunI and NheI, which removed a 2.1 kb fragmentcontaining most of the coding sequence of prtT. A 2.3 kb EcoRI/NheIfragment from pAO4-13 containing the A. oryzae pyrG gene was cloned inthe MunI and NheI sites of pEES1. The resulting plasmid, shown in FIG.4, was named pDprt. This construct was then used to transform A. nigerstrain AB4.1 to uridine prototrophy. About 150 uridine prototrophictransformants were then analyzed for protease activity on skim milkcontaining plates. Five of these did not make a halo on these platesindicating that protease activity was very low. Comparison of strainswith a disrupted prtT gene and the mutant AB1.13 strain did not show anydifferences in protease activity or phenotype.

Example 4

Overexpression of A. niger PrtT

A plasmid, pGPprt, (FIG. 5) containing the coding region and 3′noncoding sequences of prtT fused to the promoter of the A. niger gpdgene was constructed. The gpd gene codes for glyceraldehyde-3-phosphatedehydrogenase, a constitutively expressed enzyme involved in primarymetabolism. The promoter used was a fragment upstream of the codingregion.

The plasmid is transformed into A. niger AB4.1 by cotransformation withthe pyrG selection plasmid pAO4-13. Transformants with increased prtTtranscription as determined by Southern blot analysis is analysed forincreased protease expression.

Example 5

Isolation of the Zn²⁺-Finger from the A. oryzae PrtT Gene

The A. niger prtT gene is shown in SEQ ID NO: 1. The protein sequencededuced from the DNA sequence of prtT (SEQ ID NO: 2) contains a socalled Zn²⁺-finger motif expected to be responsible for the DNA bindingof the transcriptional activator encoded by prtT. The Zn²⁺-finger motifhas the following amino acid sequence: Met Thr Ala Cys His Thr Cys ArgLys Leu Lys Thr Arg Cys Asp Leu Asp Pro Arg Gly His Ala Cys Arg Arg CysLeu Ser Leu Arg Ile Asp Cys (SEQ ID NO: 34).

Degenerate primers able to code for amino acid sequences from the motifwere designed and synthesized by DNA Technology A/S, Forskerparken,Gustav Wieds vej 10, DK-8000 Aarhus C, Denmark. The primers had thefollowing sequences:

137396: A T G A C C/T G C C/T T G C/T C A C/T A C C/T T G (SEQ ID NO:35) 137397: A A/G A/G C A A/G/C/T C G A/G/C/T C G A/G C A A/G G C A/G TG (SEQ ID NO: 36)

The primers were used in a PCR reaction with genomic A. oryzae IFO4177DNA as template. The reaction was performed in a total volume of 100 μlcontaining 154 pmol of primer 137396 and 10164 pmol of primer 137397. 30PCR cycles with 56□C as annealing temperature and 30 seconds elongationtime were run. Another PCR reaction using A. niger genomic DNA and theprimers 137394: ATGACTGCCTGTCACACATG (SEQ ID NO: 37) and 137395:AGACAGCGACGGCACGCATG (SEQ ID NO: 38), which are specific for the A.niger prtT gene, was also run. In this reaction 10 pmol of each primerswas used in a 100 μl reaction. Aliquots of the two reactions wereapplied to a 3% agarose gel. After electrophoresis three approximatelyequally intense bands could be seen in the A. oryzae reaction and twobands in the A. niger reaction. One of the bands in the A. nigerreaction was more intense than the other and further had the expectedsize. One of the A. oryzae bands had the same size as the most intenseA. niger band and was isolated from the gel. The fragment was clonedinto the vector pCR2.1 (Invitrogen™). Plasmids from two individualcolonies were sequenced. The sequences are shown in FIG. 1. The twosequences differ at the end reflecting their origin in differentdegenerate primers. They are identical in the middle 40 bp, which areamplified from the genomic DNA. These 40 basepairs encode a polypeptideidentical to a part of the Zn²⁺-finger of the A. niger prtT gene.

Example 6

Isolation of the N-Terminal of the A. oryzae PrtT Gene

The inverse PCR method was used to isolate the A. oryzae prtT gene. Theprimers 144428: CACCGAGTTTTAAGCTTGCGG (SEQ ID NO: 39) and 144429:CGATCTTGATCCACGAGGG (SEQ ID NO: 40) were synthezised by DNA TechnologyA/S (Denmark). Genomic DNA was cut with a number of restriction enzymesand religated. The ligation mixtures were used as templates in PCRreactions with the primers 144428/144429. In a reaction with BamHIrestricted and religated DNA as template a fragment of approximately 2.5kb was observed after electrophoresis on an agarose gel. The fragmentwas labelled with ³²P by the random priming method and used as a probeagainst a filter containing a gridded cosmid library of genomic A.oryzae DNA. The construction of the library is described in WO 98/01470.The cosmid 11F8 showed a positive hybridization signal with the probe. ASouthern blot containing DNA from 11F8 and genomic DNA restricted withBamHI, EcoRI, PstI or XhoI was probed with the 2.5 kb inverse PCRfragment. The size of hybridizing bands from genomic DNA were comparedwith those from the cosmid DNA. Apparently some rearrangement of thecosmid had occurred since only a minority of the bands from the genomicDNA had counterparts in the cosmid. Two hybridizing fragments from thecosmid, a 1.2 kb EcoRI fragment and a 1.0 kb PstI fragment, looked equalin size to hybridizing genomic fragments. The two fragments weresubcloned from the cosmid and sequenced. Analysis of the sequence datashowed that the fragments overlap. In total 1497 bp of sequence wasobtained. Oligonucleotides encoding the Zn²⁺-finger were not containedwithin the sequence. A BamHI site was found close to one end of thesequence in a region only covered by the EcoRI sub-clone, thus allowingthe position of the sequenced genomic fragment relative to theZn²⁺-finger to be determined.

The primer 153468: CGGGATGAATTGTAGAGAGGC (SEQ ID NO: 41) was prepared byDNA Technology A/S (Denmark). The primer sequence is contained withinthe 1497 bp fragment. It is found at the end closest to the Zn²⁺-fingerand points in that direction. Two primers both of prtT Zn²⁺-fingerspecific sequence and pointing either downstream (140358) or upstream(140359) were also prepared by DNA Technology A/S (Denmark). Thesequence of the two primers are as follows: 140358:CGCAAGCTTAAAACTCGGTGCGATC (SEQ ID NO: 42) and 140359:CCTCGTGGATCAAGATCGCA (SEQ ID NO: 43). Two PCR reactions, one with theprimers 153468 and 140358 and one with 153468 and 140359, respectively,were performed with genomic DNA as template. The reaction with 153468and 140359 gave a band of approximately 1.1 kb, the other reaction gaveno visible bands, when analysed on an agarose gel. The 1.1 kb fragmentwas cloned into pCR4Blunt-TOPO (Invitrogen) and sequenced. The fragmentcontained part of the Zn²⁺-finger and overlaps with the 1497 bpfragment. Translation of the sequence showed that the region immediatelyupstream of the Zn²⁺-finger encodes a polypeptide with homology to theN-terminal of prtT from A. niger.

Example 7

Isolation of the Complete A. oryzae PrtT Gene

The remaining parts of the gene were cloned by two consecutive rounds ofinverse PCR. In the first inverse PCR reaction the genomic DNA wasrestricted with EcoRV and re-ligated. The PCR reaction was run with theprimers 175653: GATGAAAAGAATAATCGGCGAG (SEQ ID NO: 44) and 175654:CGCGGCACACTACCCCCGTTG (SEQ ID NO: 45). The reaction resulted in thesynthesis of a 1.9 kb fragment, which was cloned into the pCR4Blunt-TOPOvector and sequenced. Analysis of the sequence data showed that thefragment contains a gene with homology to the A. niger prtT gene andthat the 3′ end of the gene was missing. The second inverse PCR reactionwas thus performed. The primers were B0403G08: ATCTAGCTCAAGCATTAGCGGC(SEQ ID NO: 46) and B0403G09: AATTTCGGCCCTTTAGTGTCC (SEQ ID NO: 47).BglII restricted and re-ligated genomic DNA was used as template. A 2.4kb fragment was obtained and cloned into the pCR4Blunt-TOPO vector andsequenced. Analysis of the sequence showed that the complete A. oyzaeprtT gene had been obtained. The DNA sequence of the A. oryzae prtT geneis shown in SEQ ID NO: 48 and the deduced amino acid sequence of theencoded protein is shown in SEQ ID NO: 49.

Example 8

Disruption of the Aspergillus oryzae PrtT Gene

The A. oryzae prtT gene was disrupted using a method ofpositive/negative selection. A disruption cassette consisting of 2 kb ofthe A. oryzae prtT gene (SEQ ID NO: 48) with an insertion of the pyrGgene in the middle is cloned into a vector (pDV8) containing the herpessimplex virus I tymidine kinase gene (HSV-tk) flanked by fungalexpression signals. Expression of the tymidine kinase gene makes thehost sensitive to 5-fluoro-2-deoxyuridine. A disrupted strain can beisolated by positive selection for the pyrG gene in a pyrG minus hostand deselection of the tymidine kinase gene on 5-fluoro-2-deoxyuridine.Since the tymidinie kinase gene and the pyrG gene are present in thesame DNA fragment selection is for transformants in which a doublecross-over event has happened. The system gives fewer transformants pr.Micro g of DNA than transformation with just a disruption cassette, butthe frequency of transformants in which the desired homologousrecombination event has occurred is much higher.

The pyrG gene used here is flanked by repeats enabling a later removalby selection for 5-fluoroorotic acid resistance. The pyrG gene isisolated from the plasmid pJaL554.

The pDV8 Plasmid

pDV8 was kindly provided by Matthew S. Sachs, University of Oregon, POBox 91000, Portland, Oreg. 97291-1000, USA. pDV8 (FIG. 7) is a pSP65(Promega™) based plasmid containing the HSV-tk gene on a 1.2 kbBglII/BamHI fragment inserted between a 1.0 kb XhoI/BglII fragment ofthe A. nidulans gpd promoter and a 0.8 kb BamHI/HindIII fragmentcontaining the A. nidulans trpC transcriptional terminator.

The A. nidulans gpd promoter and the trpC transcriptional terminator aretaken from the plasmid pAN51-2 (Punt et al., (1990), Gene 93,p.101-109). The HSV-tk gene is described by McKnight S. L., (1980),Nucleic Acids Res. 8:5949-5964, Database accession no. EMBL v00470,position 252-1479. The construction of pDV8 is described inVaught-Alexander D (thesis) Expression of the herpes simplex virustype-1 thymidine kinase gene in Neurospora crassa, (1994), OregonGraduate Institute of Science & Technology, University of Portland, POBox 91000, Portland, Oreg. 97291-1000, USA. The sequence of pDV8 isincluded in this application as SEQ ID NO: 50. Single-, double- andmulticopy A. oryzae transformants of pDV8 were isolated by transforminga pDV8 derivative containing the A. oryzae niaD gene into an A. oryzaeniaD mutant. The copy number of the HSV-tk gene was determined bySouthern analysis. The transformants and the untransformed host wereinoculated onto plates containing varying concentrations of5-fluoro-2′-deoxyuridine. From inspection of the growth on the plates itwas decided to use 6 microM of 5-fluoro-2′-deoxyuridine in the platesfor future positive/negative selection experiments. At thisconcentration none of the pDV8 transformants grew, while theuntransformed host was only slightly inhibited.

Description of pJaL554

PJaL554 was constructed by ligating the 316 bp Asp718-NheI fragment tothe 5336 bp SpeI-SspBI fragment from the pyrG containing plasmid pSO2(described in WO 97/35956). Thus, pJaL554 harbours the A. oryzae pyrGgene flanked by 316 bp repeats. The construction is illustrated in FIG.8.

Construction of a PrtT Disruption Plasmid in the pDV8 Vector

A PCR reaction is performed on chromosomal A. oryzae IFO4177 DNA withthe primers B1042E05 and B1450E07.

-   B1042E05: CGCGCGTATCCTATTGCC (SEQ ID NO: 51)-   B1450E07: GCCGGAAATGTTGTACCTAC (SEQ ID NO: 52).

A fragment of 2078 basepairs is obtained and cloned into thepCR4Blunt-TOPO (Invitrogen™) vector. The resulting plasmid is sequencedwith the standard M13 forward (−40) and reverse primers to ensure thatthe correct fragment is obtained. The PCR fragment is excised from thevector by the restriction enzyme EcoRV which cuts twice internally inthe fragment. The cut sites are located at positions 1 and 1964 in SEQID NO: 48. The 1964 bp fragment is ligated with the pDV8 vector, whichhas been cut with HindIII and blunt ended by filling in the ends withthe Klenow fragment of DNA polymerase I from E. coli and dNTP. Theresulting plasmid is cut with HindIII, which is located in the prtTfragment (in the part encoding the Zn²⁺-finger) at position 962 in SEQID NO: 48, dephosphorylated and ligated with the pyrG gene isolated frompJaL554 as a 2.5 kb HindIII fragment.

Selection of prtT Disrupted Strains

The disruption plasmid described above was linearized with NotI andtransformed into A. oryzae HowB101 (described in WO 97/35956), a pyrGminus derivative of IFO4177. The transformation is done essential asdescribed in EP 0 238 023. Transformants are selected on platescontaining Coves salt solution (Cove DJ, (1966), Biochim. Biophys. Acta113:51-56), 1 M sucrose for osmotic stabilization and as carbon source,20 g/L agar, 10 mM NaNO₃ and 6 microM 5-fluoro-2-deoxyuridine (Sigma).The transformants are reisolated once on the same type of plates.Transformants carrying a disrupted prtT gene are identified by Southernblot analysis.

A strain carrying the prtT disruption is used as host for expression ofa truncated PDI gene (Protein Disulfide isomerase gene) harbored on theexpression plasmid pCaHj445 (described in U.S. Pat. No. 5,879,664).pCaHj445 is transformed into the A. oryzae prtT disrupted strain bycotransformation with the plasmid p3SR2 containing the A. nidulans amdSgene. Transformation and selection on acetamide plates is doneessentially as described in EP 0 238 023. After reisolation thetransformants are fermented in shake flasks or fermentors and the PDIprotein is purified from the fermentation broth.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An expression vector comprising: a nucleic acid construct comprising: a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49, wherein the nucleic acid sequence is operably linked to one or more control sequences, which direct the production of a polypeptide in a suitable expression host; a promotor; and transcriptional and translational stop signals.
 2. A host cell comprising the expression vector of claim
 1. 3. A host cell useful for the production of a polypeptide, wherein the host cell is a mutant of a parent fungal cell and the host cell: (a) comprises one or more DNA sequences encoding the polypeptide, (b) comprises one or more DNA sequences encoding a protease or proteases, the transcription of which is or are activated by a transcriptional activator encoded by a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is at least 95% identical with the amino acid sequence of SEQ ID NO:49; and (c) produces less of the transcriptional activator and less of the protease or proteases compared to the parent fungal cell when cultured under the same conditions.
 4. A method of producing a polypeptide, comprising: (a) cultivating the host cell of claim 3, wherein the host cell harbors a gene encoding the desired polypeptide, in a nutrient medium suitable for production of the polypeptide; and (b) recovering the polypeptide from the nutrient medium of the mutant cell.
 5. The method of claim 4, wherein the polypeptide is native to the parent cell.
 6. The method of claim 4, wherein the polypeptide is heterologous to the parent cell.
 7. The method of claim 4, wherein the polypeptide is selected from the group consisting of an antibody or portions thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or portions thereof, a regulatory protein, a structural protein, a reporter, and a transport protein.
 8. The method of claim 7, wherein the enzyme is selected from the group consisting of a hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
 9. The method of claim 8, wherein the enzyme is selected from the group consisting of an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.
 10. A host cell useful for the production of a protease, wherein the host cell is a mutant of a parent fungal cell and the host cell: (a) comprises a DNA sequence encoding the protease, the transcription of which is activated by a transcriptional activator encoded by a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is at least 95% identical with the amino acid sequence of SEQ ID NO:49, and (b) produces more of the transcriptional activator than the parent fungal cell when cultured under the same conditions.
 11. The host cell of claim 10, wherein the nucleic acid sequence encoding the transcriptional activator is operably linked to a promoter.
 12. The host cell of claim 10, wherein the desired polypeptide is an extracellular protease.
 13. A method of producing a protease, comprising: (a) cultivating the host cell of claim 10 in a nutrient medium suitable for production of the protease; and (b) recovering the protease from the nutrient medium of the mutant cell.
 14. The nucleic acid construct of claim 1, wherein the nucleic acid sequence encodes an amino acid sequence at least 97% identical with the amino acid sequence of SEQ ID NO:
 49. 15. The nucleic acid construct of claim 1, wherein the nucleic acid sequence encodes an amino acid sequence that is at least 99% identical with the amino acid sequence of SEQ ID NO:
 49. 16. The nucleic acid construct of claim 1, wherein the nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:
 49. 17. The nucleic acid construct of claim 1, wherein the nucleic acid sequence encodes an amino acid sequence consisting of SEQ ID NO:
 49. 18. The nucleic acid construct of claim 1, wherein the nucleic acid sequence is obtained from an Aspergillus cell.
 19. The nucleic acid construct of claim 18, wherein the Aspergillus cell is an Aspergillus oryzae cell.
 20. The nucleic acid construct of claim 1, wherein the nucleic acid sequence is obtained from an Aspergillus, Fusarium, Penicillium or Trichoderma cell.
 21. An isolated nucleic acid construct comprising: a nucleic acid sequence encoding an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:
 49. 22. The isolated nucleic acid construct of claim 21, wherein the nucleic acid sequence encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:
 49. 23. The isolated nucleic acid construct of claim 21, wherein the nucleic acid sequence encodes an amino acid sequence consisting of SEQ ID NO:
 49. 